Dendritic cells (DCs) are extremely potent antigen presenting cells, which play an essential physiologic role in initiating T cell responses against protein and peptide antigens. In the last 5 years, Drs. Crystal Mackall, Jay Berzofsky, and associates in the National Cancer Institute (NCI) have attempted to use autologous DCs loaded with tumor antigens in a clinical setting to stimulate host T cell responses against cancer. DCs for these studies were ?manufactured? in the Cell Processing Unit of the Department of Transfusion Medicine (DTM) by incubating peripheral blood monocytes in vitro for 4 days with recombinant GM-CSF and IL4. The resulting immature DCs were then ?matured? by incubation overnight with recombinant human CD40L. Clinical trials using DCs prepared in this manner, however, were disappointing. [unreadable] In the past year my laboratory established a collaborative relationship with the Cell Processing Unit of DTM to evaluate the function of manufactured DC products. To this end we developed a panel of assays to monitor DC phenotype, cytokine production, and migration. The panel was then used to evaluate the function of DCs prepared using the methods employed in aforementioned clinical protocols. These studies revealed several shortcomings in the clinical-grade product. CD40L-treated DCs were ineffectual in producing IL12, a cytokine crucial in promoting a T cell response, and failed to express CCR7, a chemokine receptor essential for DC migration to lymph nodes after infusion in vivo. Using the functional panel as a measure of cell function, we then modified DC culture conditions to improve the final product. We found function could be improved markedly by using recombinant gamma-interferon (g-IFN) along with CD40L to promote maturation, but cell viability and function could be improved yet further by reducing the time required for DC generation from 5 to 4 days and using a combination of endotoxin plus g-IFN in lieu of CD40L and g-IFN to promote maturation. Taken together these modification markedly increased DC yield, viability, CD83 and CCR7 surface expression and IL12 secretion. [unreadable] We also used our functional approach to compare the function of DCs generated in Teflon bags and plastic flasks. Bags have several practical advantages over flasks. In particular, they reduce the risk of microbial contamination and they do not permit cells to adhere to their surface, simplifying cell retrieval at harvest. Using our panel of functional tests however, we found that DCs grown in Teflon bags produced much less IL12 than conventional DCs in response to in vitro stimulation. Since Il12 is considered an essential element in promoting the T cell response in vivo, based on these findings, we elected not to adopt Teflon bags into our preparative procedures. We also reported our results to alert others already using Teflon for DC production to the potential functional impact of this approach. [unreadable] Having used a systematic, data-based approach for monitoring, we have substantially improved DTM DC production protocols. Consequently, DTM and NCI are in the process of initiating 3 new clinical studies using our ?second generation? DCs to stimulate T cell responses against tumor associated peptides and proteins.